Targeting proteins to the cell wall of sporulating Bacillus anthracis

Abstract

SummaryDormant spores of Bacillus anthracis germinate during host infection and their vegetative growth and dissemination precipitate anthrax disease. Upon host death, bacilli engage a developmental programme to generate infectious spores within carcasses. Hallmark of sporulation in Bacillus spp. is the formation of an asymmetric division septum between mother cell and forespore compartments. We show here that sortase C (SrtC) cleaves the LPNTA sorting signal of BasH and BasI, thereby targeting both polypeptides to the cell wall of sporulating bacilli. Sortase substrates are initially produced in different cell compartments and at different developmental stages but penultimately decorate the envelope of the maturing spore. srtC mutants appear to display no defect during the initial stages of infection and precipitate lethal anthrax disease in guinea pigs at a similar rate as wild-type B. anthracis strain Ames. Unlike wildtype bacilli, srtC mutants do not readily form spores in guinea pig tissue or sheep blood unless their vegetative forms are exposed to air.

Amide bonds assemble pili on the surface of bacilli

“…Coding sequences of the bcpA-srtD-bcpB operon were PCR amplified and cloned into pLM5 (28), to generate IPTG-inducible expression via the P spac promoter in B. anthracis. Oligonucleotides were used to create single amino acid substitutions in bcpA by site-directed mutagenesis (Table S5).…”

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

“…Plasmid DNA was purified from E. coli K1077 and used to transform B. anthracis (31). Deletion mutants were obtained by allelic replacement using the temperature-sensitive plasmid pLM4 (Table 1) (26). Briefly, 1-kb upstream and downstream DNA sequences flanking the gene of interest were PCR amplified with specific primers (Table 2), cloned by restriction digestion into pLM4, and transformed into B. anthracis.…”

“…B. anthracis Sterne 34F2 (11) and its mutants (Table 1) were cultured in brain heart infusion (BHI) broth supplemented with 0.8% NaHCO 3 . Unless otherwise indicated, cultures were incubated at 37°C or at 30°C when harboring the vector pLM4 (26). Escherichia coli strains DH5␣ (27), K1077 (dcm dam mutant) (28), or BL21(DE3) (29) were cultured in Luria-Bertani broth (LB).…”

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

“…Plasmids-The following plasmids contain B. cereus pilus genes cloned under control of the spac promoter of pLM5 (12) and were described previously: pJB39 (bcpA-srtD), pJB12 (bcpA-srtD-bcpB), pJB28 (bcpA K162A -srtD-bcpB), pJB32 (bcpAsrtD C207A -bcpB), and pJB48 (bcpA LAVAA -srtD-bcpB) (15). pJB182 encodes for bcpA-srtD-bcpB ⌬VIPNTGG719 , which contains a seven-residue deletion in BcpB, encompassing the IPNTG sorting signal motif and one residue flanking each end (VIPNTGG 719 ).…”

“…This enzyme immobilizes proteins within fully assembled cell walls, utilizing the cell wall cross-bridge as a nucleophile (11). Sortase C cuts LPNTA sorting signals and anchors proteins to the peptidoglycan cross-bridges in sporulating bacteria (12,13). Finally, sortase D catalyzes transpeptidation reactions in the assembly of pili (14,15).…”